The Di-Iron Protein YtfE Is a Nitric Oxide-Generating Nitrite Reductase Involved in the Management of Nitrosative Stress

Previously characterized nitrite reductases fall into three classes: siroheme-containing enzymes (NirBD), cytochrome c hemoproteins (NrfA and NirS), and copper-containing enzymes (NirK). We show here that the di-iron protein YtfE represents a physiologically relevant new class of nitrite reductases. Several functions have been previously proposed for YtfE, including donating iron for the repair of iron–sulfur clusters that have been damaged by nitrosative stress, releasing nitric oxide (NO) from nitrosylated iron, and reducing NO to nitrous oxide (N2O). Here, in vivo reporter assays confirmed that Escherichia coli YtfE increased cytoplasmic NO production from nitrite. Spectroscopic and mass spectrometric investigations revealed that the di-iron site of YtfE exists in a mixture of forms, including nitrosylated and nitrite-bound, when isolated from nitrite-supplemented, but not nitrate-supplemented, cultures. Addition of nitrite to di-ferrous YtfE resulted in nitrosylated YtfE and the release of NO. Kinetics of nitrite reduction were dependent on the nature of the reductant; the lowest Km, measured for the di-ferrous form, was ∼90 μM, well within the intracellular nitrite concentration range. The vicinal di-cysteine motif, located in the N-terminal domain of YtfE, was shown to function in the delivery of electrons to the di-iron center. Notably, YtfE exhibited very low NO reductase activity and was only able to act as an iron donor for reconstitution of apo-ferredoxin under conditions that damaged its di-iron center. Thus, YtfE is a high-affinity, low-capacity nitrite reductase that we propose functions to relieve nitrosative stress by acting in combination with the co-regulated NO-consuming enzymes Hmp and Hcp.

Redox cycling of the YtfE di-iron site. Limited exposure of as isolated di-ferrous YftE to air resulted in significant darkening of the sample, with an associated absorption feature at 340 nm, together with weaker absorption features at 500-520 nm (giving an orange/red color when concentrated) 2 (Fig. 3A). The absorption characteristics of the 340 nm feature, ɛ340 nm = 4.00 (±0.5) mM -1 cm -1 , were similar to those previously assigned to the mixed valent (Fe 3+ /Fe 2+ ) or di-ferric (Fe 3+ /Fe 3+ ) states of YtfE and other hemerythrin-like proteins 3,15 . Similar spectra were obtained using potassium ferricyanide (6-fold excess) or ammonium persulfate (10-fold excess) as oxidant. We note that the ~500 nm feature in hemerythrin likely originates from an oxy form (formally Fe 3+ /Fe 3+ -OOH, 2.3 mM -1 cm -1 ) or the di-ferric state (0.2 mM -1 cm -1 ). 3,6,15 As isolated, YtfE gave a featureless EPR spectrum (Fig. 3D). Exposure to air resulted in the observation of signals at g = 1.96, 1.91, and 1.88 that are characteristic of a S = ½ mixed valent, Fe 3+ /Fe 2+ di-iron center, as previously reported (Fig. 3D, Fig. S2A, Table S2). 2 Spin quantification revealed that this accounted for 37% of the YtfE protein concentration, with the remainder EPR silent. Hence, ~60% of the sample is present as di-ferric YtfE following brief (15 min) exposure to air, with the remaining ~40% in the mixed valent form. Significant changes in the CD spectrum were also observed upon exposure of diferrous YtfE to ambient O2 for 15 min, with weak features appearing at (+)320, 430, and 480 nm, and at (-)370 and 400 nm (Fig. 3B, inset, red trace).
Using non-denaturing mass spectrometry, exposure of di-ferrous YtfE in ammonium acetate to ambient O2 gave poorly resolved major peaks between 26,150 and 26,153 Da, likely corresponding to mixed valent and di-ferric YtfE along with a disulfide (full oxidation of the di-iron site and Cys30,31 would result in a -4 Da shift, see Fig. S1D and Table S1).
Anaerobic addition of dithionite to air-exposed, oxidized YftE resulted in a CD spectrum similar to that of the as isolated protein (Fig. 3B, inset, green trace), consistent with initial isolation of di-ferrous YtfE and the ability of the cofactor to undergo redox cycling. Previous studies of YtfE as an iron source for cluster reconstitution employed DTT as reductant. Addition of DTT (10 mM) to air-exposed YtfE (~100 µM diiron center) resulted in YtfE becoming colorless. Removal of low molecular weight species (<5 kDa) and re-exposure to air resulted in the reappearance of the 340 nm and 520 nm features in the absorbance spectrum, consistent with facile redox-cycling of the di-iron center, with no significant loss of iron detected (Fig. S3A). The dependence of the rate of reduction on DTT concentration was consistent with a relatively weak interaction between DTT and YtfE, see Fig. S4. The rates of reduction by 10 mM ascorbate or 0.2 mM NADH were only 20% or 10%, respectively, of that observed for DTT. Reduced glutathione (3.5 mM) was completely ineffective in reducing oxidized YtfE (Fig. S3B, C), suggesting that accessibility of the reductant to the protein/di-iron center (and not just reduction potential) is important for reduction to occur.
Direct comparison of N2O and NO production by YtfE. Nitrite (6 mM) was added to a solution of di-ferrous YtfE (437 µM) in the absence of a reductant and incubated for 15 min. Headspace gas and solution were analyzed for N2O by GC and for NO (in the form of MNIC species) by EPR spin quantification. N2O was at the lower detection limit, corresponding to 0.007 mol per mol of YtfE, while S = 3 /2 MNIC spin concentration corresponded to ~0.3 mol per mol of YtfE. This represents a lower limit of NO generated, because NO does not bind very tightly to the YtfE di-iron center and readily diffuses into solution. Indeed, a titration of di-ferrous YtfE with NO indicated that the MNIC signal did not saturate until addition of ~8 NO per YtfE (Fig. S2). YtfE-promoted formation of NO by reduction of nitrite results in oxidation of the di-iron center, to mixed valent and di-ferric forms. EPR quantification of the S= 1 /2 mixed valent form in the experiment above showed that it corresponded to ~30% of YtfE concentration. Fe 3+ has much lower affinity for NO compared to the Fe 2+ form, and so capacity to bind NO decreases as the di-iron site becomes oxidized. Furthermore, binding of NO to the mixed valent center (if this occurs) would result in a diamagnetic or integer spin, and thus EPR-silent, species.
An equivalent experiment to the above was carried out, except that air-exposed YtfE was used (~40% mixed valent form). Analysis revealed no detectable N2O, ~10% mixed valent species and MNIC corresponding to <0.01 mol per mol of YtfE. The loss of mixed valent form of YtfE demonstrates that it can react with nitrite.
Thus, overall, much higher amounts of NO compared to N2O are generated when di-ferrous YtfE reactions with nitrite, and the estimation of NO generated here is likely to be lower than the actual amount.
Bioinformatic analyses of YtfE and NO reductases. Protein BLAST searches of the NCBI RefSeq Select database for proteins similar to the E. coli K-12 YtfE, and the NO reductases Hcp, Hmp and NorV within the Enterobacteriaceae (taxid 543) were carried out. Significant alignments were taken as those with ≥35% sequence identity with ≥60% sequence coverage. This analysis yielded 90 different taxid hits for YtfE, 102 for Hcp, 110 for Hmp, and 102 for NorV. There were 11 taxid numbers that possessed YtfE but not Hcp, 15 that possessed YtfE but not Hmp, and 19 that possessed YtfE but not NorV. Of the 11 YtfE-plus taxids that lack Hcp, 7 possessed NorV. Of the remaining 4 taxids, 1 was Hmp-plus, leaving 3 taxids with an apparently orphaned YtfE (i.e. they apparently lack Hcp, Hmp and NorV). Thus, where Hcp is missing from YtfE-containing species, an alternative NO reductase is present in most cases. It is possible that an as yet uncharacterized NO reductase could be present in the few species that apparently lack any of the recognised NO reductases.
In summary, in the Enterobacteriaceae, NO production by YtfE is very often coupled to reduction to N2O (or protein nitrosation) via Hcp. Where Hcp is missing, YtfE may function together with another type of NO reductase. Overall, it is reasonable to conclude that, in general, YtfE is functionally linked to Hcp or an alternative NO reductase.   Figure S1. Additional MS and spectroscopy of YtfE. A) Native MS charge state distribution for as isolated, di-ferrous YtfE in positive mode is dominated by the monomeric form. Small amounts (~10%) of dimeric and higher order species are also observed. Deconvoluted native MS of YtfE ionized from B) ammonium formate or C) triethylammonium bicarbonate. D) Native MS confirms prolonged O2 exposure results in di-ferric and mixed valent YtfE with varying levels of disulfide bond formation, as well as damage to the di-iron site (species with mass equivalent to loss of 1Fe atom). E) Native MS of YtfE isolated from NaNO2supplemented cells, revealing a mixture of YtfE redox states and some damage to the di-iron site as well as nitrite and NO adducts. Lower intensity peaks were also observed on both the high and low mass sides.

SUPPORTING TABLES
Higher mass peaks at 26,285 and 26,308 Da corresponded to YtfE with incomplete N-terminal methionine residue removal (+131 Da) and its sodium adduct. Lower mass peaks at 26,084 and 26,114 Da corresponded to YtfE containing a single iron atom, and a possible Fe-NO species, respectively. Figure S2. EPR studies of NO-treated YtfE. A) The EPR spectrum of YtfE following exposure to NO (Fig. 3D of main paper), featuring a S = ½ mixed valent di-iron site, S = 3 /2 MNIC and S = ½ DNIC signals, was fitted using Easyspin 16 (pepper) in Matlab. Given the intrinsic linewidth of ferric EPR spectra, hyperfine coupling frequencies were not simulated. Instead, anisotropic residual linewidth was used to account for differences in line broadening. Fitting outputs are given in    Table 1 for kinetic parameters). F) CD spectra of di-ferrous Cys30Ala/Cys31Ala YtfE (46 µM) and [4Fe-4S] NsrR (20 µM) solution before (black line) and after the addition of 3 mM nitrite (red line). Only limited cluster damage was observed, consistent with a compromised auto-nitrosylation activity. Figure S6. pH dependence of YtfE-catalysed nitrite reduction. A) Initial rates analysis; fits (yellow, black, green lines) to a simple Michaelis-Menten equation, see Table 1 for full kinetic parameters. Error bars indicate standard deviation (SD) from the mean (where error bars are not visible, the SD was smaller than the data point symbol). B) pH dependence of methyl viologen-mediated, YtfE-dependent, nitrite reduction over a physiological pH range. kcat values were derived from the fits shown in A).